Glomerular disease is frequently associated with podocyte injury or effacement, characterized by flattening of podocyte foot processes. Podocyte effacement disrupts the integrity of the glomerular filtration barrier, which is thought to be the cause of proteinuria. Nephrin is a slit diaphragm protein that is critically involved in maintainin the architecture of the foot processes. Nephrin tyrosine phosphorylation appears to be an early response of podocytes to injury that leads to nephrin endocytosis and a cascade of cellular events that result in dynamic cytoskeletal modifications and ultimately cell spreading or motility in culture. These cytoskeletal modifications are also necessary for podocyte effacement in vivo. The Rho family of small GTPases, CDC42, RhoA, and Rac1, are regulators of actin cytoskeleton. Nephrin tyrosine phosphorylation has been shown to increase the activity of Rac1, resulting in reorganization of the podocyte actin cytoskeleton and increase lamellipodia. The ADP-ribosylation factor (ARF) family of Ras-related small GTPases is intricately involved in regulating endocytosis, endocytic trafficking, and recycling. ARF6, specifically, has been recognized in other cell lines for its role in trafficking and actin reorganization at the plasma membrane, possibly in coordination with Rac1. Our preliminary in vitro work shows that ARF6 is present in podocytes and plays a role in cell motility. Our working hypothesis is that nephrin tyrosine phosphorylation increases ARF6 activity which is a necessary intermediary in injury-induced podocyte effacement. To test this hypothesis, we will knockdown ARF6 in cultured mouse podocytes stably expressing nephrin and assess whether dynamic cytoskeletal modifications, such as focal adhesion turnover, which is a marker of cell migration, and ruffling/lamellipodia are observed. We will also examine potential signaling relationships between nephrin, ARF6, Rac1, focal adhesions, and ruffling/lamellipodia by immunofluorescence and immunoprecipitation. Our preliminary in vitro data suggest that increased ARF6 activity might be a key component of podocyte effacement, thus inhibition or deletion of ARF6 might protect against podocyte injury. In the second half of the proposal, using two acute podocyte injury models, protamine sulfate perfusion and nephrotoxic serum nephritis, we will assess for functional, morphological, and molecular differences between our podocyte-specific ARF6 knockout mice and control littermate. This proposal will allow us to evaluate the potential role of ARF6 as a key component of podocyte effacement in vivo which might be of therapeutic value.